Differentiation of Bone Marrow Stem Cells in Presence of Human and Xenogeneic Acellular Dermal Matrix and Collagen Membrane: an in vitro Study

Statement of the Problem: Predictable bone regeneration is an objective in implant and periodontal treatments and barrier membranes may play a significant role in osteogenic reconstruction and differentiation. Purpose: We compared the osteoblastic differentiation level of bone marrow stem cells in the vicinity of different barrier membranes. Materials and Method: In this experimental in vitro study, human collagen membrane (HCM; Regen), xenogeneic collagen membrane (XCM; Jason), human acellular dermal matrix (HADM; Regen), and xenogeneic acellular dermal matrix (XADM) were used in 4 groups. No membranes were used in the control group (5th group). Bone marrow stem cells with 150,000 cells/well density were added to the culture medium. Cellular differentiation was assessed through real-time quantitative reverse transcription polymerase chain reaction (QRT-PCR) for alkaline phosphatase (ALP) and osteopontin (OPN) gene expression, and Alizarin Red staining after 21 days. Data were analyzed using Kruskal- Wallis and Mann–Whitney statistical tests on SPSS 20 software (p Value< 0.05). Results: ALP gene expression was significantly higher in HCM group compared to other four groups (p< 0.009) followed by XADM, control, HADM and XCM groups, respectively (p< 0.001). OPN gene expression was significantly more prominent in HCM group compared to other groups (p< 0.01) followed by XADM group in which OPN gene was expressed significantly more than XCM group. OPN gene expression was not significantly different in HADM and control groups (p= 0.52). Light absorption rate was higher in HCM group compared with other groups (p< 0.012). Light absorption rate was not significantly different among HADM, XADM, and control groups (p> 0.05), though it was higher in XCM group (p= 0.009). Conclusion: Bone marrow stem cells show different levels of differentiation in the vicinity of different membranes. Generally, cell differentiation was more prominent in the vicinity of human collagen membrane.


Introduction
Guided tissue regeneration, especially in the case of bone tissue, is an accepted technique for periodontal and maxillomandibular regeneration [1], implant insertion, and repairing peri-implant lesions [2] in order to regenerate bone and gingival tissues and restore the function and aesthetics [3].
The main objective of using barrier membranes in tissue regeneration process is to prevent the growth of the gingival corium and epithelium into the lesions and maintain space for regeneration [4]. An important feature of these membranes is their ability to improve ad-hesion, cellular proliferation, and differentiation. It has been proved that the properties of barrier membrane or framework such as its composition, surface roughness, and so on can affect cellular proliferation and differentiation in regenerative therapies, though it is not completely investigated [5][6].
Various synthetic and natural membranes are introduced with appropriate results [7], including absorbable collagen membrane [8]. Since alveolar bone and periodontal ligament contain collagen, collagen membranes may have additional advantages in regenerative therapies [9]. Acellular dermal matrix (ADM) is also used in periodontal surgeries. ADM as a connective tissue substitute is associated with preventing secondary palate surgeries, shorter surgery duration, less complications and patient discomfort, no limitation in the amount of donor tissue, possibility of multiple tooth treatment in one session, natural appearance, and better patient compliance. In addition, the manufacturers claim that it improves blood circulation and fibroblast accumulation [10][11]. Despite the mentioned advantages of ADM, there are worries about ethical issues due to its human origin, which leaded to introduction of xenogeneic acellular dermal matrix (XADM). Recently, a pig-derived ADM or mucoderm is introduced as an alternative to allograft or connective tissue graft. Its advantages include lower price, xenogeneic origin, and availability in high amounts. Several studies have proved the ability of XADM to improve in vitro proliferation of human fibroblast, osteoblast, and endothelial cells [12][13][14].
An important prerequisite in regenerative and tissueengineering therapies is the presence of progenitor cells in the area and their proliferation and differentiation ability [15][16]. The mesenchymal bone marrow cells may differentiate to alveolar bone and periodontal ligaments [17]. Bone marrow-derived mesenchymal stem cells secrete the extracellular matrix, which is vital to osteogenic differentiation, and the mineralization of this matrix shows the terminal phase of osteoblast differentiation [18]. Thus, bone marrow stem cells are important in the periodontal regeneration and especially osteogenic augmentation.
Basudan et al. [19] compared the effects of absorbable collagen membrane and mucoderm in the osteogenic guided reconstruction in cranial lesions of rats. They found that the highest formation of new bone was in absorbable collagen+ xenograft and the lowest formation of new bone was in the mucoderm group. An et al. [20] concluded that pig-dermal collagen membrane might be used as a reliable membrane in the regeneration process. Pappalardo et al. [21] purposed ADM as a perfect membrane.
Since no comparative study have been conducted on effect of these barrier membranes on the level of osteoblastic differentiation in bone marrow stem cells, while histologic comparison is one of the most valuable research methods, this study intended to evaluate the abovementioned effect to provide a guide for choosing the proper membrane.

Materials and Method
This experimental in vitro study was performed on bone   Osteoblastic cell differentiation comparison was performed using AR staining which showed significant difference between five study groups in terms of light absorption (p= 0.012) ( Table 3).
Paired comparison showed that light absorption was significantly higher in HCM group compared to the oth-

Discussion
Stem cell differentiation to osteogenic osteoblast is a key part of regenerative and osteogenic augmentation therapies; thus, the level of osteoblastic differentiation of stem cells in the vicinity of four different types of barrier membranes was assessed in this study.
Duration is an important factor in the differentiation process. Often, 21 days is required for osteoblast differentiation in laboratory studies [22][23].
Osteoblastic differentiation and osteogenesis process consists of several steps. The first step is the expression of ALP and OPN genes. Then, these genes will lead to protein production. Afterwards, bone cells should get mature to secrete extracellular matrix. Finally, the most important function of a bone cell is its ability to secrete extracellular matrix and calcium deposit [18]. Since the XCM used in this study (Jason) contained a high amount of type III collagen based on manufacturer claim [24], it may not bring the advantages of HCM containing type I collagen. This may be a probable factor of superiority of HCM over XCM.
The results in study groups indicated that differentiation level based on OPN gene expression was similar to ALP gene expression. OPN gene expression was higher in the vicinity of HCM followed by mucoderm, which shows the role of these membranes on gene expression during differentiation process.
In this study, collagen membrane and ADM were evaluated due to their high consumption and extended use in periodontal treatments. Moreover, XADM was also assessed because of its novelty and limited related studies.
It is well proved that cellular adhesion, proliferation, Miron et al. [29] assessed the proliferation and differentiation of human osteoblasts in the vicinity of collagen membranes combined with bone morphogenic protein 2 (BMP2) and transforming growth factor beta1 (TGFβ1).
Adherence of osteoblasts to all these membranes and improvement of osteoblasts' proliferation parameters compared to control group was observed. In addition, PCR analysis showed that BMP2 increased osteoblast differentiation markers. Moreover, based on AR staining, BMP2 leaded to improved mineralization of primary osteoblasts compared to control and TGFβ1 groups.
Kobayashi et al. [30] investigated the role of collagen membrane (bioguide) coated with bone conditioned media (extracted from cortical bone of pig mandible) on adhesion and differentiation of osteoblasts via real-time PCR and AR staining. They reported increased expression of alkaline phosphatase, osteocalcin, osteogenic sialoprotein, and increased AR staining intensity.

Conclusion
The results of the current study suggest different behavior of bone marrow stem cells in osteoblastic differentiation process in the vicinity of different membranes.
Osteoblastic differentiation and ontogenesis processes occurred significantly better in the presence of HCM compared to other membranes.